The long-term objective of this research is a detailed understanding of herpesvirus DNA polymerases and the drugs that target them. These enzymes, which include a catalytic subunit (Pol) and an accessory (processivity) subunit that stimulates long-chain DNA synthesis, are prototypes for family B polymerases that include human replicative DNA polymerases. Herpesvirus DNA polymerases are also excellent targets for antiviral drugs. This latter property is especially important to human health, as we do not fully understand how the leading drugs against herpes simplex virus (HSV) and human cytomegalovirus (HCMV), acyclovir and ganciclovir, respectively, selectively inhibit the corresponding viral polymerase. Moreover, new drugs are urgently needed for treatment of herpesvirus infections, particularly HSV and HCMV infections resistant to current drugs. In this application, unanswered questions regarding the polymerase subunits and drugs that target these proteins are addressed. Specific aim 1 is to solve structures of the HSV DNA polymerase bound to primer-template. Structures of the enzyme bound to primer-template terminated with a dideoxynucleoside plus a deoxynucleoside triphosphate (dNTP) will be determined by cryo-electron microscopy (cryo-EM), X-ray crystallography, or both. Subsequent structures will include complexes with acyclovir at the primer-terminus or acyclovir triphosphate as a mimic of the dNTP. These structures will provide information regarding multiple features of the polymerase, including conformational changes that occur upon DNA binding, the relationship of the polymerase active site to the exonuclease active site, how acyclovir is recognized by the enzyme, how specific substitutions confer resistance, and how the HSV processivity subunit, UL42, interacts with DNA to bind tightly yet be able to diffuse by hopping. To explore how the polymerase interacts with other HSV replication proteins, cryo-EM studies of replication fork complexes will be undertaken. These studies will address numerous unanswered questions regarding the arrangement of replication proteins at the fork, and the still-unknown structure of the primase-helicase. Specific aim 2 is to solve structures of the HCMV polymerase bound to primer-template, again using cryo-EM, X-ray crystallography, or both. In this aim, one focus will be on understanding how primer-templates containing ganciclovir in the primer induce chain termination of the polymerase, particularly whether ganciclovir distorts the primer-template backbone and thus its interactions with the polymerase and exonuclease domains. A second focus will be on how the HCMV processivity subunit, UL44, interacts with DNA when it is part of the polymerase holoenzyme. In particular, the structures should resolve whether the protein binds to DNA primarily through residues in a cavity resembling that of the human processivity factor, PCNA, or whether it binds primarily through flexible loop residues. In both aims, structural information regarding how the herpesvirus polymerases differ from their host counterparts may abet drug discovery.